Steric trapping reveals a cooperativity network in the intramembrane protease GlpG.

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins

Native Proteomics in Discovery Mode Using Size-Exclusion Chromatography–Capillary Zone Electrophoresis–Tandem Mass Spectrometry

Native proteomics aims to characterize complex proteomes under native conditions and ultimately produces a full picture of endogenous protein complexes in cells. It requires novel analytical platforms for high-resolution and liquid-phase separation of protein complexes prior to native mass spectrometry (MS) and MS/MS. In this work, size exclusion chromatography (SEC)-capillary zone electrophoresis (CZE)-MS/MS was developed for native proteomics in discovery mode, resulting in the identification of 144 proteins, 672 proteoforms, and 23 protein complexes from the Escherichia coli proteome. The protein complexes include four protein homodimers, 16 protein-metal complexes, two protein-[2Fe-2S] complexes, and one protein-glutamine complex. Half of them have not been reported in the literature. This work represents the first example of online liquid-phase separation-MS/MS for characterization of a complex proteome under the native condition, offering the proteomics community an efficient and simple platform for native proteomics

Membrane Depth-Dependent Energetic Contribution of the Tryptophan Side Chain to the Stability of Integral Membrane Proteins

Lipid solvation provides the primary driving force for the insertion and folding of integral membrane proteins. Although the structure of the lipid bilayer is often simplified as a central hydrophobic core sandwiched between two hydrophilic interfacial regions, the complexity of the liquid-crystalline bilayer structure and the gradient of water molecules across the bilayer fine-tune the energetic contributions of individual amino acid residues to the stability of membrane proteins at different depths of the bilayer. The tryptophan side chain is particularly interesting because despite its widely recognized role in anchoring membrane proteins in lipid bilayers, there is little consensus about its hydrophobicity among various experimentally determined hydrophobicity scales. Here we investigated how lipid-facing tryptophan residues located at different depths in the bilayer contribute to the stability of integral membrane proteins using outer membrane protein A (OmpA) as a model. We replaced all lipid-contacting residues of the first transmembrane β-strand of OmpA with alanines and individually incorporated tryptophans in these positions along the strand. By measuring the thermodynamic stability of these proteins, we found that OmpA is slightly more stable when tryptophans are placed in the center of the bilayer and that it is somewhat destabilized as tryptophans approach the interfacial region. However, this trend may be partially reversed when a moderate concentration of urea rather than water is taken as the reference state. The measured stability profiles are driven by similar profiles of the <i>m</i>-value, a parameter that reflects the shielding of hydrophobic surface area from water. Our results indicate that knowledge of the free energy level of the protein’s unfolded reference state is important for quantitatively assessing the stability of membrane proteins, which may explain differences in observed profiles between <i>in vivo</i> and <i>in vitro</i> scales

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH

ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380–550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems